Why are not all chilies hot? A trade-off limits pungency

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Why are not all chilies hot? A trade-off limits pungency
David C. Haak, Leslie A. McGinnis, Douglas J. Levey and Joshua J. Tewksbury
Proc. R. Soc. B published online 21 December 2011
doi: 10.1098/rspb.2011.2091
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Proc. R. Soc. B
doi:10.1098/rspb.2011.2091
Published online
Why are not all chilies hot? A trade-off
limits pungency
David C. Haak1,*, Leslie A. McGinnis1, Douglas J. Levey2
and Joshua J. Tewksbury1
1
Department of Biology, University of Washington, PO Box 351800, 24 Kincaid Hall, Seattle,
WA 98195-1800, USA
2
Department of Biology, University of Florida, PO Box 118525, Gainesville, FL 32611-8525, USA
Evolutionary biologists increasingly recognize that evolution can be constrained by trade-offs, yet our understanding of how and when such constraints are manifested and whether they restrict adaptive divergence in
populations remains limited. Here, we show that spatial heterogeneity in moisture maintains a polymorphism
for pungency (heat) among natural populations of wild chilies (Capsicum chacoense) because traits influencing
water-use efficiency are functionally integrated with traits controlling pungency (the production of capsaicinoids). Pungent and non-pungent chilies occur along a cline in moisture that spans their native range in
Bolivia, and the proportion of pungent plants in populations increases with greater moisture availability. In
high moisture environments, pungency is beneficial because capsaicinoids protect the fruit from pathogenic
fungi, and is not costly because pungent and non-pungent chilies grown in well-watered conditions produce
equal numbers of seeds. In low moisture environments, pungency is less beneficial as the risk of fungal infection is lower, and carries a significant cost because, under drought stress, seed production in pungent chilies is
reduced by 50 per cent relative to non-pungent plants grown in identical conditions. This large difference in
seed production under water-stressed (WS) conditions explains the existence of populations dominated by
non-pungent plants, and appears to result from a genetic correlation between pungency and stomatal density:
non-pungent plants, segregating from intra-population crosses, exhibit significantly lower stomatal density
(p ¼ 0.003), thereby reducing gas exchange under WS conditions. These results demonstrate the importance
of trait integration in constraining adaptive divergence among populations.
Keywords: genetic correlation; secondary compounds; defence; capsaicinoids;
capsaicin; natural selection
1. INTRODUCTION
Environmental heterogeneity drives spatial variation in selection and has long been assumed to play a key role in the
maintenance of phenotypic variation by creating and enhancing adaptive trade-offs under divergent selection [1–5].
The basis for this assumption is that natural selection acts
on whole organisms rather than isolated traits and, therefore,
adaptation may often be constrained by trade-offs among
traits that interact to determine individual phenotypes (i.e.
functionally integrated traits [1,5]). Adaptive trade-offs are
a cornerstone of evolutionary ecology [6–8], frequently
explaining the maintenance of polymorphisms at range
margins [9] and along environmental gradients [10,11].
Nonetheless, direct evidence of adaptive trade-offs is surprisingly rare [5] and most often represented by studies of
experimental evolution in laboratory conditions [5,12,13].
Evidence of adaptive trade-offs in natural populations is lacking or surprisingly equivocal [6]; patterns attributed to
fitness trade-offs resulting from environmental differences
can often be attributed to alternative mechanisms [5].
Thus, despite many examples of trait polymorphisms,
our understanding of the mechanism(s) underlying the
maintenance of phenotypic variation among natural
populations remains largely theoretical [14,15].
A central challenge in documenting adaptive trade-offs
in natural populations is finding a species in which phenotypic variation is clearly tied to fitness consequences that
change along a well-defined environmental gradient [16]
and in which benefits and functionally linked costs of
the phenotype are readily apparent [7]. Variation in the
expression of constitutive chemical defences in plants has
been suggested as a tractable area in which to examine
these issues, as ecological benefits are easily measured
and physiological costs appear unavoidable [17]. In practice, however, the benefits of chemical defence are often
well documented, but the costs are often elusive [18,19].
One reason for the paucity of data on costs associated
with a given plant defensive trait is that most defensive
traits belong to an integrated suite of traits, which function
concomitantly or even synergistically [20,21]. Such interactions make it difficult to ascribe fitness differences
among phenotypes to a particular trait. Another limitation
has been the focus on plant defensive traits related to herbivory. Although the measurement of herbivory itself is
straightforward, the relationship between herbivory and
fitness is complex and variable [22].
Here, we focus on a plant defensive trait that avoids these
pitfalls: pungency in wild chili peppers (Capsicum spp.). Pungency in wild chilies has a simple genetic basis [23] and is
expressed only in the fruit, where it acts to protect seeds
*Author and address for correspondence: Department of Biology,
Indiana University, Bloomington, IN 47405, USA (dhaak@
indiana.edu).
Electronic supplementary material is available at http://dx.doi.org/
10.1098/rspb.2011.2091 or via http://rspb.royalsocietypublishing.org.
Received 5 October 2011
Accepted 30 November 2011
1
This journal is q 2011 The Royal Society
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2 D. C. Haak et al.
Adaptive trade-off limits pungency
rainfall
(cm yr–1)
latitude (°)
(a) –19.0
110
–19.5
100
–20.0
90
80
–20.5
70
–21.0
60
–21.5
50
–22.0
40
–64.0
proportion of pungent plants
(b)
–63.5
–63.0
longitude (°)
–62.5
–62.0
1.0
0.8
0.6
0.4
0.2
60
70
80
annual rainfall (cm yr–1)
90
Figure 1. (a) The natural rainfall gradient across southeastern Bolivia where polymorphic populations occur; pie charts indicate
the proportions of pungent plants. (b) The proportion of pungent plants in each population increases as a function of precipitation (r 2 ¼ 0.83, F1,19 ¼ 100.5, p 0.001). Triangles (black, 29%; grey, 45%; white, 88%) indicate the study populations.
[24], thereby providing the direct link to changes in fitness
that is often missing from studies of chemical defence
and herbivory [22]. Pungency is also polymorphic in some
wild chili species, and multiple polymorphic populations
have been identified along natural environmental gradients
[24,25]. These attributes make the wild chili pepper an
excellent system in which to investigate adaptive constraints.
Chili peppers get their ‘heat’, or pungency, from
capsaicinoids, and two capsaicinoids, capsaicin and dihydrocapsaicin, account for more than 95 per cent of this
pungency [26]. Pungency appears to be a synapomorphy,
as basal chilies (Capsicum cilliatum) lack pungency and
most derived chilies (e.g. Capsicum annuum) are all pungent [27]. Southeastern Bolivia is the putative centre of
diversity for wild chilies; at least three Capsicum species
co-occur and are polymorphic in pungency, with plants
producing either pungent or non-pungent chilies growing
side by side [25]. A pathogenic and pervasive fungus
(Fusarium spp.) produces strong selection for pungency
in at least one of these species, Capsicum chacoense [4].
In particular, capsaicin appears to protect chili fruits
and seeds from Fusarium attack. It also protects chili
fruits from consumption by granivorous rodents without
reducing consumption by seed-dispersing birds [28,29].
Given these well-documented benefits of capsaicin, we
ask: is the observed polymorphism in wild chilies
Proc. R. Soc. B
maintained by a cost of pungency? In other words, ‘why
are not all chilies hot?’ To answer this question, we
focus on the evolutionary ecology of pungent and nonpungent forms of C. chacoense along a natural moisture
gradient in southeastern Bolivia.
2. MATERIAL AND METHODS
(a) Pungency and populations
Natural populations of C. chacoense are either polymorphic
for pungency (hot or not) or monomorphic (all hot). In
addition, these populations vary in the degree of pungency
among pungent plants [25]. We have examined variation in
pungency and the polymorphism for pungency in 21 populations along a 300 km transect in southeastern Bolivia that
co-occurs with a gradient in moisture (figure 1a). In the
dry northeast, populations are 15–20% pungent and the percentage of pungent plants increases towards the wetter
southwest, where populations are 100 per cent pungent (in
7 census years, we have not located a non-pungent chili in
these populations; figure 1a and [25]). We have censused
each of the focal populations over 5 years (between 2002
and 2009). To determine new recruits, we tagged plants
each year and counted only new seedlings. We analysed
changes in the proportion of pungent plants within locations
through time by comparing model fit for a series of
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Generalized Linear Mixed Models with binomial error distribution using R [30]. All models included location as a
random effect. We tested the significance of pungency proportion with location, year and location by year as fixed
effects. Model fits were estimated using maximum likelihood
(ML) with a logit-link function. Pungency was determined
by tasting in the field and verified by high-performance
liquid chromatography as in Tewksbury et al. [25].
(b) Field sampling and growing conditions
We selected three polymorphic populations that span the
range in rainfall, pungency (per cent of population pungent)
and plant density found in polymorphic C. chacoense populations ([25] and figure 1b; electronic supplementary
material, table S1). We collected fruits from pungent and
non-pungent plants in each population, and seeds from
these fruits were grown in the University of Washington glasshouse. We selected 11 maternal lineages from each pungency
by population class for a total of 66 lineages, forming a population of 330 plants. These maternal plants were grown in the
University of Washington glasshouse, and selfed fruits were
selected to form a common garden population.
(c) Water stress experiment
All plants were grown under identical well-watered (WW)
conditions in the University of Washington glasshouse,
until after the first flower in each treatment group was
observed, at which point, plants from within each lineage
were assigned to one of two treatments and individually
watered: a WW treatment (mimicking, as much as possible,
the average rainfall during fruiting and flowering seasons in
our WW site (electronic supplementary material) or a
water-stressed treatment (WS; developed to mimic the average rainfall data from the driest location; electronic
supplementary material). Under natural conditions, water
is not limiting until fruit set, and fruit set has been described
as the period in which chili peppers are most susceptible to
drought stress [31]. We imposed the water stress treatment
after the first flower in each treatment group was observed
to replicate the natural water availability and ameliorate the
effects of unnatural water stress on overall growth (electronic
supplementary material). Data were analysed using a generalized linear model with lineage, treatment and pungency,
and all interactions as fixed effects and a logit link transformation. Stepwise reduction [32] yielded a final model retaining
the treatment by pungency interaction effect.
(d) Stomatal density
To isolate the relationship between pungency and stomatal
density, we developed an artificial population segregating
for pungency by reciprocally crossing two populations of
pungent non-pungent parents and analysing stomatal density from 30 plants in the F2 population. Stomatal density is
influenced by plant and leaf age and by environmental conditions. To control for these effects, we standardized the
watering regime (using the WW watering treatment, §2c),
and age-matched all plants based on two age criteria:
(i) date of germination and (ii) uniform height of 60 cm. Epidermal peels were obtained from sets of four leaves collected
at 10, 25 and 50 cm. Stomata were visualized using a Zeiss
microscope at 400. Stomatal density was estimated by averaging the count of abaxial stomata in the visual field (using a
reticle scale corrected for magnification) of four regions for
each peel. There were no differences in the non-independent
estimates of stomatal density along the height of the plant
Proc. R. Soc. B
seed production (no. per plant)
Adaptive trade-off limits pungency
D. C. Haak et al.
3
500
200
**
100
well-watered
water-stressesd
treatment
Figure 2. The total seed output for pungent (open) and
non-pungent (filled) plants under well-watered and waterstressed conditions. Error bars are +1 s.e., n ¼ 352, GLM,
pungency treatment p ¼ 0.005.
(a rough surrogate for leaf age). These averaged data were
analysed using a t-test to detect mean differences in stomatal
density between pungency classes. The pungency of each
plant was verified by tasting and liquid chromatography.
3. RESULTS
Populations of wild chilies in Bolivia vary considerably
in the proportion of pungent plants in the population
(figure 1; electronic supplementary material, table S1),
consistent with previous observations [25]. Measuring
the frequency of pungent plants among new recruits over
7 years in these populations revealed no significant shifts
in the proportion of pungent plants across the cline
(electronic supplementary material, table S1). Variation
in total annual rainfall predicts this stable cline in pungency
(figure 1b, r 2 ¼ 0.83, F1,19 ¼ 100.5, p 0.001). Under
restricted water conditions in the glasshouse, pungent
plants produced 50 per cent fewer seeds per plant than
non-pungent plants, but under WW conditions, fruit production was nearly identical (figure 2; GLM binomial
error distribution, pungency environment interaction
F ¼ 10.3, p ¼ 0.005, n ¼ 352).
In these populations, pungent and non-pungent plants
co-occur (within 1 m2) and are completely inter-fertile
(within and between populations) [23]. Because water-use
efficiency differences can result from differences in leaf morphology [33], we investigated morphological differences
between pungent and non-pungent plants. For plants
grown in the glasshouse, from field-collected seed, the
only detected leaf morphological feature differing between
types within population was stomatal density. To establish
a mechanistic link between pungency and stomatal density,
we constructed populations segregating for pungency
and examined stomatal density. In these populations, pungent plants exhibited 40 per cent greater stomatal density
than non-pungent plants; t-test, t ¼ 23.7474, d.f. ¼ 7.7,
p-value ¼ 0.006, n ¼ 19 (figure 3).
4. DISCUSSION
In Southeast Bolivia, C. chacoense populations are distributed from high-rainfall regions in the southwest to the
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4 D. C. Haak et al.
Adaptive trade-off limits pungency
stomatal density (no. mm–2)
8
7
6
5
4
non-pungent
pungent
Figure 3. Stomatal density (number of stomata per square
millimetre) segregates with pungency (n ¼ 19, t-test, p ¼
0.006) in the F2 generation of populations generated from
crossing pungent by non-pungent parents.
dry chaco regions north and east, and they are subject to
multiple biotic and abiotic selective pressures that vary in
importance across this space and among years. Physiological trade-offs appear to mediate the relative
advantage of pungent versus non-pungent phenotypes
across this cline. Non-pungent plants from our polymorphic C. chacoense populations show clear evidence of
adaptation to water-limited conditions; non-pungent
plants, which have significantly lower stomatal densities
on their leaves than pungent plants, produce twice as
many seeds under water stress (figure 2). These plants
are disproportionately found in dryer areas (figure 1a),
where water stress is more common and the fitness advantage of pungency is lower, owing to a reduction in the
frequency of insect and fungal attack on chili fruit [4].
The positive correlation between rainfall and the proportion of pungent plants within a population (figure 1b)
suggests a fitness trade-off between protection from
fungal attack (chemical defence) and costs of producing
capsaicin in drier environments (figure 1b). It is possible
that some frequency-dependent effect on pungency might
alone maintain the observed gradient, e.g. variation in
Fusarium abundance or infection (vector abundance), but
this is unlikely, as Tewksbury et al. [24] found that more
than 90 per cent of the fruit from all 12 populations
were infected with Fusarium. Given the ubiquity of these
fungal pathogens, we would expect pungency to sweep
through all of the populations as even a moderate infection
level significantly impacts seed viability [24]. This suggests
that an integrated response to biotic and abiotic selective
pressures maintains this polymorphism in pungency.
A potential mechanism underlying this proposed
trade-off in resource allocation is that non-pungent
plants use limited water resources more efficiently
through fixed differences in stomatal density. Evidence
from cultivated chilies suggests a negative relationship
between stomatal density and water-use efficiency [34].
Additionally, empirical data from diverse taxa support a
negative functional relationship between water-use efficiency and stomatal density [35]. In this study, using
plants segregating for pungency, non-pungent plants
exhibited a 40 per cent lower abaxial stomatal density
than pungent plants (figure 3). This suggests that
Proc. R. Soc. B
pungent plants have a lower water-use efficiency and consequently suffer a fitness cost under limited water
conditions. This finding is consistent with other studies
that have reported trade-offs in the production of defensive compounds under limited resource conditions [36].
The putative genetic correlation between stomatal density
and pungency shows some degree of overlap in the full
range of recombinants. Thus, while we failed to detect a
recombinant pungent phenotype that overlapped the
mean stomatal density of non-pungent plants, it is not
clear from these data whether this genetic correlation is
sufficient to constrain pungency in drier environments
on its own. A plausible alternative is that pungency is constrained by the combination of the genetic correlation
between pungency and stomatal density and relaxed
selection on pungency [24].
The genetic correlation with stomatal density could be
the result of tight linkage or pleiotropy. Although our data
do not allow us to distinguish between these mechanisms,
two lines of evidence suggest pleiotropy. First, our focal
populations tend to be in Hardy – Weinberg equilibrium
(D. C. Haak 2011, unpublished data) for presumably
neutral microsatellite loci. Second, production of capsaicin (pungency) and lignin rely on a shared biochemical
pathway, and pungent plants show increased seed coat
lignin deposition per seed at the cost of overall seed production under limited water conditions (D. C. Haak
2011, unpublished data), functionally coupling wateruse efficiency and pungency. Studies dissecting shared
quantitative trait loci (QTL) have found pleiotropic
genes, linking, for example, flowering time and wateruse efficiency in Arabidopsis [37]. Similarly, an Arabidopsis
transcription factor controls epidermal patterning and has
pleiotropic effects on seed coat deposition [38]. Nonetheless, general inferences of pleiotropy from shared QTL
are not warranted; while many correlated traits share
QTL, a recent review has found that few of these are
pleiotropic [39]. Future work will elucidate the genetic
mechanism underlying the relationship between pungency and stomatal density.
Natural selection is constrained by the relationship
between populations and their environments as well as
by interactions among traits within organisms [40].
Although we do not know whether the genetic correlation
between pungency and stomatal density is a result of linkage disequilibrium or pleiotropy, the result is ecological
specialization, in which high performance in one environment carries a significant cost in other environments [7].
In contrasting environments, pungent and non-pungent
chilies from the same population display fitness differences clearly attributable to changes at the pungency
locus and perhaps its influence on stomatal density
(a proxy for water-use efficiency). Thus, we propose
that pungent C. chacoense plants are limited from adapting
to the driest regions in this cline by a trade-off with wateruse efficiency, thus explaining the gradient in proportion
of pungent plants (figure 1a). More generally, stomatal
density and pungency trade-off to limit the evolution of
pungency, supporting a central tenet of evolutionary ecology theory—that divergent natural selection can drive
population differentiation [41].
We are grateful to the University of Washington glasshouse
staff, D. Ewing, P. Beamon, J. Milne and E. Forbush for
Downloaded from rspb.royalsocietypublishing.org on January 25, 2012
Adaptive trade-off limits pungency
care of the plants, and R. Nariya for many hours at the
microscope. We also thank H. D. Bradshaw, B. E. Miner,
K. Brady and M. Krosby for helpful discussion. Climate
data used in this paper were produced with the Giovanni
online data system, developed and maintained by the
NASA GES DISC. D.C.H. was supported by a Graduate
Research Fellowship from the National Science
Foundation, J.J.T. by a National
Geographic Grant,
D.C.H. by a grant-in-aide from Sigma Xi and D.C.H. by a
Giles Botanical Field Research Award from the University
of Washington Department of Biology.
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